Project Summary/Abstract High-throughput screening (HTS) is a powerful method for the discovery of new drug leads for target enzymes. In HTS assays, the activity of the target enzyme is often evaluated by quantifying a small-molecule or cofactor that is produced or consumed by the enzyme. While antibodies are a mainstay of small-molecule detection as- says, they do have severe limitations, which are largely tied the challenge of functionalizing small molecule targets without masking key functional groups. As a result, conjugation of the target molecule to a carrier pro- tein for antibody generation is laborious and often decreases the specificity of the antibodies generated. Addi- tionally, HTS assays that use small-molecule binding antibodies require that a labeled version of the target be produced to act as a competitor in the assay. We propose that DNA structure switching (SS) biosensors can overcome these limitations, as these sensors provide a direct fluorescence readout upon target binding, and do not require that the target be covalently labeled. Additionally, these biosensors utilize nucleic acid ap- tamers, which can be generated using in vitro selection methods that do not necessarily require that the target be modified or immobilized. While SS biosensors hold tremendous potential for use in HTS and other small- molecule detection assays, the currently available protocols for generating these biosensors are time- consuming and at times unreliable. Thus, we propose an improved method for the in vitro selection of SS bio- sensors that is anticipated to provide more efficient enrichment of functional sequences. This will both reduce the time required and increase the success rate for generating biosensors to new small-molecule targets of interest. In Aim 1, we will develop and implement this selection method to generate a SS biosensor for Coen- zyme A (CoA). In Aim 2, we will utilize this biosensor to produce a fluorescence polarization HTS assay for CoA, the product of histone acetyltransferases (HATs). The immediate impact of this research will be an im- proved HTS assay for screening HATs, which will address a significant unmet need in drug discovery for a number of epigenetic diseases including neurological disorders, cancers, and cardiovascular disease. From a broader perspective, this research will provide a rapid and reliable method for generating SS biosensors for small-molecule targets, which will accelerate development of HTS assays for diverse enzyme drug targets.